The diagnosis of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years. In addition to traditional histological staining techniques and immunohistochemical assays, in situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans. Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.
Chromogenic alkaline phosphatase (AP) detection on tissue has commonly been performed by a combination of a substituted naphthol AS phosphate and a diazonium salt. This methodology was introduced by Burstone in the late 1950's. See Burstone, J. Histochem. Cytochem. 6 322-39 (1958). The technique involves the liberation of naphthol by AP, which then reacts with a diazonium salt to generate an insoluble azo dye that precipitates at the epitope site.
Chromogenic techniques have been limited due to a limited number of chromogenic substrates that impart different colors. Accordingly, what is needed in the art are chromogenic substrates that provide new colors for use in immunohistochemical and hybridization assays. The addition of novel chromogenic substrates provides for, for example, the multiplexing of different targets that are identified by different color deposits, all of which are identifiable on a single tissue on a single slide.